这项工作是由鲁昂大学学院研究与创新在生物医学（IRIB），诺曼底的细胞成像平台（PRIMACEN），INSERM，IBiSA，欧洲区域发展基金支持（ERDF - PeReNE，区域间4A），在LARC - 神经科学网和区域上诺曼底。

动物（男性或女性Wistar大鼠）出生和成长在一个认可的动物基金（批准B.76-451-04），根据用于护理和使用实验动物的指导法。实验授权调查员（MB，DV和LG）按照欧洲共同体理事会指令（9月22日2010/63 / UE，2010）和农业的法国文化部的监督下进行。 1.准备媒体和工具<ol><li>在生物安全柜，在无菌水从含有氯化钙 （1.85克/升）/加入MgSO 4（0.9767克/升）无MgCl 2的10倍的原液制备1×Hank氏BSS（HBSS）中。添加碳酸氢钠 （350微克/毫升）1倍的HBSS溶液。 </li><li>在生物安全柜中，添加N2添加剂（来自100×储备液）和青霉素（100单位/ ml）-streptomycin（0.1毫克/毫升）溶液到Dulbecco改进的Eagle培养基（DMEM）的营养组合TURE的F-12（1:1）。 </li><li>在无菌条件下，制备的胞质荧光染料称为细胞追踪格林在DMSO中的等分试样（25微升，2毫摩尔）（1.075毫升1毫克）。在一个15毫升的锥形试管稀释在5ml的DMEM中的一个等分试样。 </li><li>准备填补冰桶保持媒体在4℃。 </li><li>净化实验室台面和工具用70％的乙醇。 </li></ol>从P10大鼠2解剖小脑<ol><li>迅速斩首幼鼠（P10）与后面耳朵弯曲操作剪刀为了得到脊髓的开头。 </li><li>在被斩首的头部的背面，使从颈部到鼻子与精细虹膜剪皮肤正中切口，分离从头骨的皮肤细虹膜剪刀和杜蒙＃3钳。 </li><li>用细虹膜剪刀精心制作从基两侧切口头骨延髓区域。有两个＃3镊子取出颅骨解剖。取下文胸在从使用相同镊子颅骨任何粘附。 </li><li>转移用含2毫升冰冷的HBSS介质的抹刀培养皿（直径35毫摩尔）的勺子端脑。 </li><li>把培养皿含有大脑在一个较大的陪替氏培养皿（直径100毫米）充满冰和转移到立体显微镜的阶段。 </li><li>在立体显微镜下，用两个＃3镊子dilaceration隔离从脑小脑。同样地，去除残留脊髓和脑膜膜。 </li><li>转移在一个新的HBSS填充培养皿小脑（直径35毫米，2.5毫升）用刮刀的勺子端，并保持在冰上。 </li></ol> 3.准备急性小脑切片<ol><li>下的立体显微镜，切开蚓部和右半球之间的小脑由两个磁头所指示箭头在图2A与标准解剖刀手柄＃3固体和＃15手术刀。 </li><li>把一滴青色oacrylate上胶vibratome标本盘，并等待15-25秒，以消除有毒溶剂蒸汽。 </li><li>收集切小脑用锅铲的勺子端和去除多余的HBSS用干净的纸巾。 </li><li>把小脑接近样品盘。修复切口边缘到样品盘，并等待10秒。 </li><li>插入试样盘进入缓冲器托盘操纵器，并且将其旋转以便小脑的横向轴线垂直于所述刀架。修复标本盘内六角扳手，并填写轻轻缓冲液盘与HBSS中，直到小脑被覆盖。 </li><li>负载碎冰到冷却浴中。 </li><li>清洁刀片三次，用70％的乙醇，以消除任何油。 </li><li>将刀片插入刀架并用固定螺丝固定。 </li><li>放置标本的后缘后面的刀刃右（从用户的观点）和将其定义为起始点。使用前进命令将t定义他结束试样的前边缘后点。 </li><li>选择切片速度为2.5和切片频率8.选择修剪厚度为180微米。开始组织切片。 </li><li>使用大孔玻璃截断巴斯德吸管，并转移到HBSS含培养皿（直径35 MM）拾取各部分保持在冰上。 </li><li>使用两个＃5镊子，小心翼翼地从与小脑叶片干扰时删除脑膜。收集最多的5片每小脑（ 图2B，C）。 </li><li>取下小脑切片脑膜仔细立体显微镜下的两个＃5镊子和分离小叶轻轻更好的探头负载。 </li></ol>生活Interneurons 4.荧光染色<ol><li>用一个大孔玻璃截断巴斯德吸管转移小脑切片到6孔板（最大3片/每孔）。吸出HBSS中。 </li><li>孵育在5毫升升片（3最大值）荧光染料oading溶液（10μM）。 </li><li>避光，用铝箔覆盖的微孔板。把它放在一个陀螺移动台以35rpm的速度进行10分钟，在室温以促进细胞标记。 </li><li>在Transwell小的膜转印片插入（3.0微米孔径; 图2D）具有一个大孔玻璃截断巴斯德吸管。吸吸管与加载介质。 </li><li>取出插入并填写好1.9的10ml DMEM。更换刀片和对切片以覆盖组织的顶部添加100μl的DMEM中。 </li><li>将包含在培养室中培养插入物（37℃，5％CO 2）2小时，其足以在ML观察选区的板。骗组织平，以允许附接在插入膜（ 图2E）。确保片不干燥。 </li></ol> 5. 离体成像共聚焦肉眼<ol><li>转板无塑料盖成一个孵化器连接到一个共焦macroscope的立场。放置一个玻璃盖的macroscope的嵌入板。保持腔室的温度保持在37.0℃±0.5℃，并用恒定气流（95％O 2，5％的CO 2）通过板插入以保持pH值恒定供给片。等待时间推移实验前2个额外的人力资源。 </li><li>为了显现的GC迁移中的组织切片，通过配有一个X2干物镜共焦激光扫描macroscope照亮制备具有488纳米波长的光通过激光二极管的装置（工作距离:38毫米，直径58毫米，NA = 0.234），并检测从500到530nm的荧光发射。 <ol><li>精细地解决选区的移动，获取图像为1.5至2.0的附加光学放大因子。在单个焦平面或高达沿z轴10不同的焦平面的每30分钟达12小时收集GNS的图像。 </li></ol></l我&gt; <li>必要时，拆下玻璃盖，加入DMEM的生物激活剂或抑制剂的小卷（1-10微升）与10微升吸管研究其对GC迁徙的影响。 </li></ol> 6.细胞跟踪<ol><li>为电影的每一次，通过在ImageJ的所述ecart型模式执行Z堆叠凸起。调制对比度和连续的图像的亮度水平，以方便识别和标记的糖皮质激素的跟踪。地图上的快照引用（=在t 0）手动每个位置。 <ol><li>使用"手动跟踪"插件在分析粒子菜单，确定通过点击在时间推移每个单元体的重力点。导出电子表格中的原始跟踪数据。 </li></ol></li><li>从重组ImageJ的导出原始跟踪数据的智能自制程序（http://primacen.fr，用PHP编写的代码）标识每个单元和相关职务。使用该程序，CALCULATe本总行驶距离，并为每个细胞迁移的平均速率。分类和使用在相同的程序相应的过滤器在控制和治疗条件比较细胞迁移的特性。 </li></ol>

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作者什么都没有透露。

本协议描述的在Transwell小系统和GC的带有绿色荧光染料通过共聚焦宏观上出生后发育过程中，研究细胞迁移荧光标记急性P10大鼠小脑切片文化。该协议允许细胞迁移的观察一段长达12小时，并调控因子中的迁移，包括在实验期间激动剂或神经肽拮抗剂，酶抑制剂，细胞信号传导调节剂或有毒物质的可能作用的测试。一个小孔用枪头插入膜是必要的，以促进化合物的管理在孵化中。自制的弯曲尖吸移管可以用来促进溶液的输送。 对活组织切片的细胞​​迁移的研究的一个问题是，该组织本身的运动可以使细胞追踪困难。而先前的方法提出轻轻稳定SLICES用尼龙网或薄层鼠尾胶原7,17的，这种技术的主要优点是6孔培养板的含有从CO 2培养箱下客观上膜插入小脑切片的简单和直接转印的共焦macroscope。集成温度和CO 2控制器还提供了细胞迁移9种必需适当的和恒定的环境参数。因此，培养条件中的观察和组织运动保持被最小化，因为片是公附着到膜的插入。组织的稳定性是通过以下的切片边缘或浦肯野细胞应采集过程中保持固定的引用的位置进行验证。另外，分布在6个孔的板的小脑切片（12至18）可以快速地详细观察电动载物台和光学变焦。由于干客观，外延的大量工作距离（X2，39个MM）-observation是自由浸没化合物和施用在培养基中容易得多。因此，在二氧化碳培养箱和共聚焦环境参数和文化支持宏观上的相似性导致对生物样品的最大保护。 协议的另一个优点是大视场，因此在大量的细胞可同时观测到的。例如，我们先前已经决定，荧光地方选区的径向迁移的ML密度为1124±138细胞/毫米2 18。共焦肉眼检查（X2，NA = 0.234）具有较低的横向分辨率相比共聚焦显微镜（40X，NA = 1.25），但细胞的GC体可以容易地跟踪和迁移的平均速率是可比之间两个技术接近7,18 。 除了技术改进图像采集，组织切片和TH的质量贴标电子质量是关键点的成功试验。在解剖过程中始终保持媒体和组织上的冰，消除油vibratome刀片和不利用组织切片与胶水接触。矢状面和横切面都适于分别径向和切向偏移。使用温育不同长度在小脑的不同皮质层适当的检测。长孵育时间（最多8小时）是必要的检测无数个地方选区的迁移在PCL和IGL。自GC迁移是在特定的时空窗生理过程，阳性对照的是，细胞具有迁移正常。特别是，众多的主轴GC在ML是矢状小脑切片的主要健康指标之一。对于启动实验，观察GC运动在ML的建议。事实上，气相色谱与垂直细长细胞体的形状应被视为一个参考点开始交流quisition与连续控制（2小时）和处理（2小时），可以在对ML容易地进行周期。 荧光染料，如细胞跟踪家庭或通过基因构建体表达的荧光蛋白可以用作示踪剂细胞迁移的研究。由于气相色谱迁移（1堆每30分钟）的慢动力学，多色实验也可以在连续的模式，因为4激光器光束（405，488，532和633纳米）来进行被在系统上可用。考虑到向心力和离心力放射状迁移，跟踪其他interneurons也可以实现18。特别是，更小许多细胞类型都可以更容易地本地化具有大视场。最后，这个协议可以用于研究细胞迁移在小脑发育的其他阶段，还包括其他脑区。

在显影小脑，八种不同类型的神经元产生的第二胚胎周和第二周产后在啮齿类1之间顺序。始发最初从，伯生发区，未成熟粒细胞（GC）将被从外部颗粒层（EGL），仲生发区2产生的最后一个神经元。在前三产后周，小脑皮层是有组织的四层包括EGL，分子层（ML）的一个面理化结构，所述浦肯野细胞层（PCL）和内颗粒层（IGL）（ 图1）。通过心的迁移，不成熟的地方选区，谷氨酸能interneurons，在大约2天到达的IGL。由第三产后一周，EGL消失，IGL构成所谓的颗粒层（GL），在成人小脑。在GL，GCS收到苔藓纤维和单极刷细胞的兴奋性突触输入，抑制性突触投入高尔基细胞轴突。在ML，GC轴突使兴奋性突触与GABA能神经元，包括浦肯野细胞，篮状细胞，星形细胞和细胞高尔基2。 从出生后早期啮齿类动物获得的急性小脑片实时观察细胞的运动表明，糖皮质激素改变它们的形状伴随其小脑皮层3路线变化时迁移的方式和速度。在头两个产后周，GC前体积极增殖在EGL的顶部。在EGL的中间部分，有丝分裂后的GC在其过程中较大的切向的方向迁移。在EGL-ML边界，GCS减缓它们的运动，细胞开始进入一个短的垂直下降过程到ML。在对ML，选区有一个垂直伸长的细胞体，一个薄拖尾处理和更大体积主导过程，且径向迁移沿着贝格曼胶质纤维。在里面PCL，GCS停止他们的运动，但长时间的固定相（2小时）后，他们越过PCL-IGL边界。在IGL，选区移向在没有神经胶质纤维支持层的底部。一旦领先的工艺的尖端接近IGL-白质（WM）的边界，GCS缓慢停止移动。小脑的横截面优选用于东瀛切向迁移的研究，而矢状切片致力于放射状迁移的ML，PCL和IGL。 GC是运动的一些监管因素，包括神经肽（ 如生长抑素，PACAP）已经确定，但迄今为止参与GC移民在每个皮质层中的时空控制的完整的机制仍然知之甚少1,4,5,6。 GC迁移一直在通过视频和共聚焦显微镜在过去的20年使用或者透射光照明孤立培养的细胞或荧光检测研究ACUTE小脑切片。最初的亲脂性的DiI，以及最近的"单元格追踪"染料和细胞表达的荧光蛋白被用于共焦或双光子显微镜7,8。成功的实验取决于许多的，使协议简单但不容易的具体程序。特别是，急性切片有观测期间被稳定一般用自制尼龙网状网络9。的光照明的强度必须是尽可能低，以避免光毒性和漂白所提议的多点扫描共聚焦显微镜方法。此外，温度和CO 2，因为不稳定的主要环境参数可影响神经细胞迁移。为了方便和细化实验程序，我们已开发了一种共聚焦肉眼检查协议，限制片的动作，可确保恒定的环境参数，减少了光漂白，增大视场和consequ（milimeters的量级）ently细胞（数十），可以通过图像分析跟踪的数量。因此，180微米厚的切片上膜插入培养和6-孔板下一个商业共聚焦macroscope装备大孵育室，温度和CO 2的控制器和一振动控制系统的一个2X机动目标直接传送。时的失误和z栈然后进行几个小时和药理学工具或生物活性分子可以被添加或在孵化介质递送。这种方法也可以适用于研究的其他类型的不同发育阶段的小脑或大脑的神经元的迁移。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s modified eagle medium (DMEM) nutrient mixture F-12</td>
			<td>Sigma-Aldrich</td>
			<td>D8437</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution 10X</td>
			<td>Sigma-Aldrich</td>
			<td>H1641</td>
			<td></td>
		</tr>
		<tr>
			<td>PACAP38</td>
			<td>INRS, Canada</td>
			<td></td>
			<td>Bourgault et al., 200919</td>
		</tr>
		<tr>
			<td>PAI-1</td>
			<td>Calbiochem</td>
			<td>528208</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Fisher Scientific / Gibco/ invitrogen</td>
			<td>O973</td>
			<td></td>
		</tr>
		<tr>
			<td>cyanoacrylate glue</td>
			<td>Loctite</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Tracker Green CMFDA</td>
			<td>Invitrogen</td>
			<td>C2925</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyester Transwell-Clear inserts</td>
			<td>Corning</td>
			<td>3452</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture cluster</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher Scientific</td>
			<td>BP231-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture dish 35 mm diameter</td>
			<td>BD Falcon</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture dish 100 mm diameter</td>
			<td>Thermo SCIENTIFIC</td>
			<td>130182</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylen tube (15 ml)</td>
			<td>BD Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>Fisher Chemical</td>
			<td>E/0800DF/21</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>biological safety cabinet fume hood</td>
			<td>Thermo Scientific</td>
			<td>MSC9 Class II A2</td>
			<td></td>
		</tr>
		<tr>
			<td>adjustable-volume pipette (0,5-10 &#181;L)</td>
			<td>Eppendorf</td>
			<td>4910 000.018</td>
			<td></td>
		</tr>
		<tr>
			<td>gyro-rocker, SSL3</td>
			<td>Stuart</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator, Hera Cell 150</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>vibrating blade microtome, VT1000S</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>confocal macroscope, TCS LSI</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>temperature controller</td>
			<td>PeCon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2-controller</td>
			<td>PeCon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope, M205 C</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Operating scissors, curved, blunt/blunt</td>
			<td>Medicon</td>
			<td>03.03.17</td>
			<td></td>
		</tr>
		<tr>
			<td>Hardened fine iris scissors, straight, sharp/sharp</td>
			<td>FST</td>
			<td>149090-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #3 and #5 forceps</td>
			<td>FST</td>
			<td>11293-00 and 11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome injector blades/single edge</td>
			<td>Leica Microsystems</td>
			<td>39053250</td>
			<td></td>
		</tr>
		<tr>
			<td>standard scalpel handle #3 solid</td>
			<td>FST</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical blade #15</td>
			<td>Swann-Morton</td>
			<td>205</td>
			<td></td>
		</tr>
	</tbody>
</table>

ex vivo imaging of postnatal cerebellar granule cell migration using confocal macroscopy

在出生后的发育未成熟粒细胞（兴奋性）表现外颗粒层中的分子层和浦肯野细胞层切迁移，然后放射状迁移到达小脑皮层内部颗粒层。默认情况下，迁徙过程中诱导细胞死亡或神经元的错位，导致小脑不同功能的赤字。心颗粒细胞迁移涉及几种机制，如趋化性和细胞外基质降解，引导细胞向它们的最终位置，但被调节在每个皮层细胞迁移的因素仅部分已知的。在我们的方法中，急性小脑切片从P10的大鼠制备的颗粒细胞被标记有荧光细胞质标记和组织进行培养上膜插入的4至10小时，起始细胞迁移的实时监测通过共聚焦肉眼检查在37℃之前在里面CO 2存在的。在他们在小脑的不同皮质层迁移，颗粒细胞可以暴露于神经肽激动剂或拮抗剂，蛋白酶抑制剂，细胞内效应甚至有毒物质如醇或甲基汞阻滞剂，调查在神经细胞迁移的调节他们的可能作用。

在出生后早期小脑，选区呈现在他们的模式和迁移的速度显著变化，因为它们跨越不同皮质层1（ 图1）。本节说明可以通过研究GC移民在他们的自然细胞环境将得到的结果的例子。标记有绿色荧光染料P10大鼠小脑组织切片检查下一个共焦macroscope（ 图3A）和我们表明，糖皮质激素在ML径向迁移为18微米/小时的平均速度（ 图3B，C）。迄今为止，相互作用的作用/神经元和神经胶质细胞包括调节因子和参与细胞迁移的控制在各皮层分子机制之间的通信在很大程度上是未知的。因此，主要的问题是确定的神经肽，神经递质，神经营养因子和细胞外基质成分，可以在这些皮质层特定发挥作用速度在其迁移过程FIC变化。垂体腺苷酸环化酶激活多肽（PACAP）主要是检测到的PCL，而且在ML和IGL在头两个产后周啮齿类7,10,11。导致了对ML在GC的79％的速度下降的PACAP38（10 -6 M）的培养基中的应用。例如，在对ML选区的移动速度从11.9微米/小时的PACAP38（ 图4A）给药后丢弃在控制条件下，以2.5微米/小时。组织型纤溶酶原激活物（tPA）是蛋白水解级联，导致细胞外基质（EM）的成分的降解，例如细胞粘附分子或层粘连蛋白12,13中的一员。 tPA和纤溶酶原，tPA的的基板，在皮质层产后小脑14,15,16的开发过程中进行检测。 PAI-1（10 -7 M），内源性的tPA的抑制剂，施用降低78％GC的迁移的ML。例如，糖皮质激素从19.2微米/小时降低迁移速度在ML在控制条件下，以4.2微米/小时此外，PAI-1（ 图4B）之后。这些结果表明，PACAP上施加的GC运动和该丝氨酸蛋白酶的tPA方便的GC在显影大鼠小脑的ML迁移直接抑制作用。 <img alt="图1" src="/files/ftp_upload/52810/52810fig1.jpg" /> 图1:GC移民在产后小脑皮质的3D表示。 1-4，扩展在EGL GC过程和切向偏移。5，在ML径向迁移沿着贝格曼胶质纤维6中，瞬态固定相在PCL 7中，胶质无关径向在IGL迁移。8，完成EGL;在IGL GC GC迁移，颗粒细胞，在红色。，外颗粒层; B，贝格曼神经胶质细胞，在暗紫色; G，高尔基体细胞，在黄色; CF，攀登纤维，蓝色; 克 ，postmigratory颗粒细胞，在浅绿色; IGL，内部颗粒层; MFT，苔藓纤维终端，深绿色; ML，分子层; P，浦肯野细胞，在浅紫色; PCL，浦肯野细胞层。这个数字已经被修改5。 <img alt="图2" src="/files/ftp_upload/52810/52810fig2.jpg" /> 图2:P10小脑切片体外培养（A）从P10老鼠小脑解剖。比例尺= 6毫米。 （ 二）显微住180微米厚的切片小脑通过立体显微镜的。比例尺= 3毫米。 （C）在较高的倍率，小脑四个皮质层（EGL，ML，PCL，IGL）输出已区别的。比例尺= 1毫米。 （D）的荧光标记后，组织切片放置在培养插入物（24毫米直径）到6孔板中。 （ 五）文化与插入组织培养处理的聚酯膜的示意图。 <a href="https://www.jove.com/files/ftp_upload/52810/52810fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52810/52810fig3.jpg" /> 图3:在大鼠P10小脑皮质层选区的动态迁移 P10大鼠小脑片，其中地方选区都标有绿细胞质荧光染料（A）Macroconfocal视图（XYZ，2D投影）。比例尺= 75微米。 （ 二）时间推移成像显示GC运动中的ML共聚焦宏观上在控制条件下4小时。星号（*）符号标志着GC SOMA。时间（以分钟）被指示在每个显微照片的底部。比例尺= 10微米。 （ 三）通过GC SOMA移动的距离连续变化。 <img alt="图4" src="/files/ftp_upload/52810/52810fig4.jpg" /> 图4:气相色谱迁移的神经肽和蛋白酶抑制剂的影响 （A） 的气相色谱跟踪对ML通过共聚焦肉眼检查2小时在控制条件下，然后进行2小时的垂体腺苷酸环化酶激活多肽（PACAP）的存在。 （B）的气相色谱跟踪对ML通过共聚焦肉眼检查2小时在控制条件下，然后进行2小时的纤溶酶原激活物抑制剂-1（PAI-1）的存在。

Watch this Scientific Journal Video about Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy at JoVE.com

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

产后的小脑颗粒细胞迁移利用共聚焦肉眼离体成像

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

ex-vivo-imaging-postnatal-cerebellar-granule-cell-migration-using

Research

JoVE Journal

Neuroscience

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

9.3K 次观看.  Inserm, IRIB, University of Rouen. During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

视频: Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

科研

神经科学

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

体外实时成像在小鼠神经上皮单细胞分裂

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

成神经细胞迁移的小鼠急性脑片在体内电穿孔产后和时间推移成像

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

小脑轴突的激光Nanosurgery<em&gt;在体内</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering the molecular signals that cooperatively guide neuronal migration in the embryonic brain is therefore important to understand how the complex neural networks form which later support postnatal life. Facial branchiomotor (FBM) neurons in the mouse embryo hindbrain migrate from rhombomere (r) 4 caudally to form the paired facial nuclei in the r6-derived region of the hindbrain. Here we provide a detailed protocol for wholemount ex vivo culture of mouse embryo hindbrains suitable to investigate the signaling pathways that regulate FBM migration. In this method, hindbrains of E11.5 mouse embryos are dissected and cultured in an open book preparation on cell culture inserts for 24 hr. During this time, FBM neurons migrate caudally towards r6 and can be exposed to function-blocking antibodies and small molecules in the culture media or heparin beads loaded with recombinant proteins to examine roles for signaling pathways implicated in guiding neuronal migration.

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the neural tube, but migrate to reach appropriate targets. Facial branchiomotor (FBM) neurons are a useful model to study neuronal migration. This protocol describes the wholemount ex vivo culture of mouse embryo hindbrains to investigate mechanisms that regulate FBM migration.

鼠标后脑<em&gt;前体内</em&gt;企业文化来研究面部Branchiomotor神经元迁移

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering ...

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

小胶质细胞的小脑颗粒细胞使用L-亮氨酸甲酯选择性耗尽

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

使用荧光素二乙酸酯 - 碘化丙啶双重染色在小脑颗粒神经元培养中评估神经元活力

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...